DTI Drug Target Insights 2024; 18: 84-93 ISSN 1177-3928 | DOI: 10.33393/dti.2024.3169 ORIGINAL RESEARCH ARTICLE Drug Target Insights - ISSN 1177-3928 - www.aboutscience.eu/dti © 2024 The Authors. This article is published by AboutScience and licensed under Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0). Commercial use is not permitted and is subject to Publisher’s permissions. Full information is available at www.aboutscience.eu Cytotoxic activity, selectivity, and clonogenicity of fruits and resins of Saudi medicinal plants against human liver adenocarcinoma Ali Hendi Alghamdi 1, Aimun A.E. Ahmed 2,3, Mahadi Bashir 1, Haidar Abdalgadir4, Asaad Khalid 5, Mohamed E. Abdallah6, Riyad Almaimani 6, Bassem Refaat 7, Ashraf N. Abdalla 8,9 1Surgery Department, Faculty of Medicine, Al-Baha University, Al Baha - Saudi Arabia 2Pharmacology Department, Faculty of Medicine, Al-Baha University, Al Baha - Saudi Arabia 3Pharmacology Department, Faculty of Pharmacy, Omdurman Islamic University, Khartoum - Sudan 4Biology Department, Faculty of Science, Al-Baha University, Al Baha - Saudi Arabia 5Substance Abuse and Toxicology Research Center, Jazan University, Jazan - Saudi Arabia 6Department of Biochemistry, Faculty of Medicine, Umm Al-Qura University, Makkah - Saudi Arabia 7Laboratory Medicine Department, Faculty of Applied Medical Sciences, Umm Al-Qura University, Makkah - Saudi Arabia 8Department of Pharmacology and Toxicology, College of Pharmacy, Umm Al-Qura University, Makkah - Saudi Arabia 9Department of Pharmacology and Toxicology, Medicinal and Aromatic Plants Research Institute, National Center for Research, Khartoum - Sudan ABSTRACT Background: Edible fruits and resins provide various benefits to mankind including potential medicinal applica- tions. This study aimed to determine the cytotoxicity, selectivity, and clonogenicity of fruits and exudates of certain Saudi medicinal plants (Anethum graveolens (BEP-09), Opuntia ficus-indica (L.) Miller (BEP-10), Boswellia serrata Roxb. ex Colebr. (BEP-11), and Commiphora myrrha (BEP-12)) against human liver adenocarcinoma (HepG2). Methods: Initial cytotoxicity and cell line selectivity against different cell lines were screened using MTT assay. The most promising extract was subjected to gas chromatography-mass spectrometry (GC-MS) analysis to deter- mine the main phytoconstituents. Clonogenicity was checked for the most active extract. Results: The selected plants’ fruits and resins possess a significant cytotoxic activity estimated as IC50. The fruit of BEP-10 was found to be the most active extract against liver cancer cells (IC50 = 2.82) comparable to both doxo- rubicin (IC50 = 1.40) and camptothecin (IC50 = 1.11). It showed a selectivity index of 4.47 compared to the normal human foetal lung fibroblast (MRC5) cells. BEP-10 showed a dose-dependent clonogenic effect against HepG2 cells comparable to the effect of doxorubicin. The GC-MS chromatogram of BEP-10 extract revealed the presence of eight small polar molecules, representing 73% of the total identified compounds and the rest three molecules (27%) were non-polar constituents. The furan derivatives represent the chief components in BEP-10 (16.3%), while the aldehyde 5-(hydroxymethyl)-2-furancarboxaldehyde was found to be the main molecule (13.2%). Conclusion: The fruits of BEP-10 have a potential cytotoxic effect particularly against HepG2. The identified phy- toconstituents in the tested plant extract might contribute to the investigated cytotoxic activity. Keywords: Clonogenicity, Cytotoxicity, MTT assay, Opuntia ficus-indica, Saudi plants, Selectivity index Received: June 16, 2024 Accepted: September 18, 2024 Published online: October 22, 2024 Corresponding author: Ali Hendi Alghamdi email: drahendi2030@gmail.com are used as flavouring agents. It is an erect, robust, and rather glabrous annual aromatic herb. The leaves are three to four pinnate, with the ultimate segments narrowly linear to filiform. The flowers are yellow, and appear in umbels, with an elliptic cremocarp. It has been recognized in differ- ent systems of traditional medicine for the treatment of dif- ferent diseases and ailments of humans. The plant is used as an antispasmodic, carminative, and anti-inflammatory. It is also used as medicine for loss of appetite, cough and cold, menstrual cramps, liver problems, oral care, strengthening the immune system, protection against bone degradation, and urinary tract disorders (1). The antioxidant and antican- cer activities of A. graveolens were investigated in human, Introduction Anethum graveolens L. is a member of the Apiaceae fam- ily locally known as Shabat-sanout. This plant has a long his- tory of use as a spice in our food, where its seeds and leaves https://doi.org/10.33393/dti.2024.3169 https://creativecommons.org/licenses/by-nc/4.0/legalcode https://orcid.org/0000-0003-4451-3820 https://orcid.org/0000-0002-6403-132X https://orcid.org/0009-0004-4114-1253 https://orcid.org/0000-0001-8734-2588 https://orcid.org/0000-0001-7944-2597 https://orcid.org/0000-0003-4267-1016 https://orcid.org/0000-0003-4770-9319 Alghamdi et al Drug Target Insights 2024; 18: 85 © 2024 The Authors. Published by AboutScience - www.aboutscience.eu lung, breast, and cervical carcinoma cell lines (2-4). Nam et al (5) studied the anti-inflammatory and protective properties of A. graveolens (dill seeds) on oesophageal mucosal dam- age in rats induced by reflux esophagitis and revealed good physiological activity and the possibility of being used as a medicinal, food, and functional resource for the prevention and therapy of gastro-oesophageal disorders. A systematic review and meta-analysis of randomized controlled trials investigated the effects of A. graveolens (dill) supplementa- tion on lipid profile and glycaemic control, showing that A. graveolens could provide favourable effects on insulin resis- tance and serum low-density lipoprotein (6). The anthelmin- tic action of A. graveolens essential oil was found to be a promising alternative in the control of sheep gastrointestinal nematodes (7). Khare (8) reported that it was used for eye problems. Opuntia ficus-indica (L.) Miller is a member of the family of Cactaceae locally known as ElBarshoumy–El TeenElShawki. It is a shrub or arborescent. Leaves are subulate and deciduous. Fruits are ellipsoidal or obovoid, red, yellow to orange, fleshy, edible. The plant is widely distributed in the south and the southwest of Saudi Arabia. It is widely known for its benefi- cial properties (9). Historically it was used as food for humans and farm animals and in folk medicine due to its nutritional properties and beneficial activities (10). Traditional medicine has used many plant extracts for human and animal wellness, due to their beneficial properties in wound healing and skin. In this regard, the study of Trombetta et al (11) is most helpful. Traditionally it was used as a treatment for gastritis, hyper- glycaemia, hypercholesterolaemia, arteriosclerosis, diabetes, and prostatic hypertrophy, and it also has hypolipidaemic action and immune regulation function in the gastrointestinal tract (12). The protective properties of various plant extracts on airway inflammation related to exposure to PM10 and die- sel exhaust particles were evaluated in mice (13). The antioxi- dants of O. ficus-indica as important inhibitors of free radical formation were reported by Castañeda-Arriaga et al (14), as well as antioxidants and inhibition of the sugar digestive enzyme activities of polyphenols by in vitro experiments (15). The powder of peel and seed of the plant efficiently removes the aqueous manganese cations (16). The gums were used to improve the quality of breads and cakes (17). The phenolic phytoconstituents, antioxidant and antiacetylcholinesterase activities of O. ficus-indica peel and flower teas were evalu- ated after in vitro gastrointestinal digestion (18). It modulates the intestinal microbiome in obese women and improves host metabolism (19). Polysaccharides from O. ficus-indica showed a regulating effect on intestinal flora of cyclophosphamide- induced immunosuppressed mice by effectively increasing the white blood cell count index and improving their thymus and spleen, while effectively promoting the secretion of inter- leukin (IL)-4, IL-1beta, tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma (20). Indicaxanthin isolated from fruits enhances glucose dysmetabolism and reduces insulin resistance in mice fed the high-fat diet (21). Boswellia serrata Roxb. ex Colebr. belongs to the Burseraceae family locally known as luban-Kundur. These are moderate to large deciduous trees. They have papery flakes of bark and “yellowish green resin” inside. Leaves are compound and alternate. Flowers are white and are distrib- uted in southern Saudi Arabia. The extract of B. serrata exhibited a potential effect in protecting the intestinal epithelium compared to lipopoly- saccharide (LPS)-stimulated cells (22,23). The diuretic activ- ity of gum extract in albino rats was investigated (24,25) and significant diuretic, kaliuretic, and natriuretic effects were observed. Synergistic antimicrobial activity of essential oil from B. serrata was studied with various azoles against azole- resistant strains of Candida albicans pathogens (26). The plant was used as a culture medium for micropropagation and as a natural source of nonsteroidal anti-inflammatory and antiarthritic agents (27). The antianaphylactic and mast cell stabilizing effects of boswellic acid have been assessed on passive paw anaphylaxis and revealed potential immuno- modulatory activity (28). Recently, Boswellia spp. and its iso- lated bioactive phytoconstituents were traditionally used to treat chronic disease, inflammation, oral health, and micro- bial infection (29). Gum is traditionally used for the treatment of various inflammations that affect the skin, gums, eye, gas- trointestinal tract (GIT) in addition to respiratory inflamma- tion disorders such as bronchitis, asthma, laryngitis, etc. (30). Commiphora myrrha (Nees) Engl. belongs to the Burseraceae family, locally known as El Murr Elihejazi. These are spiny, deciduous, almost shrub or small tree, with short thorns, producing a hard translucent yellowish gum resin. Leaves are green to greyish or glaucous, variable in shape, and minute in size. Native to Saudi Arabia, the plant is tradi- tionally used as an anti-inflammatory and in the treatment of infectious diseases, making it a very popular and valuable alternative and traditional medicine (31,32). It was found to heal wounds, ulcers, and various diseases of the pulmonary, GIT, and urinary system (33). Furanodienone and curzerene are bioactive compo- nents detected in the oil of the resinous exudate of C. myr- rha that were tested and found to influence the spread of viruses by intervening at different stages of the virus life cycle (34). The antiosteoporotic effects of C. myrrha and its polysaccharide were inhibited through osteoclastogenesis (35). Sesquiterpenoids and its phytoconstituents isolated from the resinous exudate of C. myrrha were found to inhibit the migration of human hepatocellular liver carcinoma cells (HepG2) according to a dose-dependent pattern (36). A pilot study revealed that C. myrrha has significant analgesic prop- erties (37). A combination of herbs (Commiphora mukul, C. myrrha, and Terminalia chebula) functions as an antioxidant, hypolipidemic, and antidiabetic substance; it could be recom- mended as a helpful herbal remedy for those with diabetes (38). The ethanolic extract of the resin of C. myrrha showed anti-obesity potential (39). It showed a hepatoprotective effect against D-GalN/LPS-induced liver injury in a rat model through multiple pathways (40). Murr (C. myrrha) is benefi- cial in treating eye diseases, as kahl forms in ulcers of the eye with other drugs. In Unani medicine, Murr is applied as a mixture with aabe mooli (radish juice) to eyes for cataracts, where the eyes are cleaned after dissolving murr in milk and in infraorbital haemorrhage (41-44). Tumour-related destructive autoimmune responses can affect the eye, where autoantibody-mediated destruction In vitro cytotoxicity of Saudi medicinal plants86 © 2024 The Authors. Drug Target Insights - ISSN 1177-3928 - www.aboutscience.eu/dti of retinal cells is induced by ectopic expression of periph- eral tumour-related ocular antigens (45). Neuroendocrine tumours can metastasize to the orbits of the eyes of the midgut carcinoid (46). In the Philippines, the majority of con- junctival, eyelid, and orbit tumours were benign, and retino- blastoma was the most prevalent type of intraocular tumour, while the majority of them were malignant (47). An update is needed to reorient the way to predict the prognosis of pae- diatric cancers, such as rhabdomyosarcoma and retinoblas- toma, and also adult cancers, such as uveal melanoma and lymphomas, and the benefit of targeted therapies, immuno- therapy, or even chemotherapy (48). Fruits and resins are usually used as nutritional supple- ments and are rarely used for medical purposes. Attempts are being made to look for the constituents of the plant that can prevent and reverse cancer. In this study, in vitro anti- cancer activity and cell line selectivity of two fruits and two resins were studied in three different cell lines, while clono- genicity was investigated against HepG2. Furthermore, the most promising extract was subjected to gas chromatogra- phy-mass spectrometry (GC-MS) analysis to determine the main active phytoconstituent(s). Materials and methods Phytochemical studies Identification of plant materials Four plants – A. graveolens (fruit, coded as BEP-09), O. ficus-indica (fruit, BEP-10), B. serrata (resin, BEP-11), and C. myrrha (resin, BEP-12) (Fig. 1) – were identified and taxo- nomically classified by an expert taxonomist (Dr. Mohamed, HAA, Department of Biology, Faculty of Sciences, Al-Baha University) and were compared to herbarium materials and different volumes of the flora of Saudi Arabia (49-51). Voucher herbarium specimen numbers (BUH-76,77,78, and 79) were deposited at the Department of Biology of the Faculty of Science of Al-Baha University. FIGURE 1 - Opuntia ficus-indica (L.) Miller (BEP-10) grows in the Al-Baha area, KSA. Collection and extraction of plant materials Plant specimens were collected from different sites in Baljurashi province (Wadi El khaitan), Al-Baha area, in April 2021. Fruits (1 kg) and resins (1 kg) were shade-dried and then powdered using a mechanical grinder. The dried materi- als were macerated in 80% ethanol v/v) for 1 week at room temperature. The resulting residues were filtered, pooled, and evaporated to dryness to provide viscous green to brown- ish syrups. The crude extracts, so obtained, were transferred to a Petri plate, allowed to dry, and finally weighed. The percentage of yield was calculated using the formula: yield% = (Afforded extract weight)/(Air-dried weight) × 100. The plants yielded extracts weighing 1.53, 1.73, 1.62, and 2.17 g, respectively. GC-MS analysis The dried fruits of O. ficus-indica (L.) Miller (BEP-10) were dissolved in methanol to reach a concentration of 1 mg/mL and diluted 1:10 v/v in methanol (100 μg/mL). The diluted sample was analysed using a GC-MS instrument (Thermo Scientific, USA) attached to a trace ultra-GC and ISQ detector and an AS 3000 autosampler. The separation of components was carried out using a TR-5MS column (Thermo Scientific, USA) with a length of 30 cm, a diameter of 0.25 mm, and a film thickness of 0.25 mm. Helium was used as a carrier gas at 1.2 mL/min with constant flow. The injection port was set at 32°C for 5 minutes, followed by a ramp to 205°C at a rate of 5°C/min and a hold time of 5 minutes. This was followed by a ramp to 280°C at a rate of 5°C/min and hold time of 5 minutes and at the end to 300°C at a rate of 5°C/min and a hold time of 5 minutes. The maximum oven temperature was set at 320°C. A volume of 2 μL diluted extract was injected into the system in split mode with the mass spectrometer run in electron ionization mode with 0.6 scan periods throughout the mass range of 60-900 amu (minutes). Both the tempera- ture of the MS ion source and the transfer line were adjusted to 320°C and 350°C, respectively, using a 1 kV electron mul- tiplier voltage. Identification of phytoconstituents Xcalibur software was used for mass spectral data analy- sis and the fragmentation patterns of each constituent were matched with MS data in the instrument database and built-in libraries including MAINLIB, NIST, and REPLIB. The phytocom- pounds present in the extract were identified by comparing them with the structures available in the computer library, and the percent abundance of each component was deter- mined using the peak area as reference. The reported bio- logical properties of the detected compounds are based on data from Duke’s Phytochemical and Ethnobotanical Database (52). Cancer cell studies Cancer cell culture In this study, three cancer cell lines, MCF7 (human breast adenocarcinoma), HT29 (human colorectal adenocarcinoma), Alghamdi et al Drug Target Insights 2024; 18: 87 © 2024 The Authors. Published by AboutScience - www.aboutscience.eu and HepG2 (human liver adenocarcinoma), were used, in addition to MRC5 (normal human foetal lung fibroblast), all were from American Type Culture Collection (ATCC), USA. Three cancer cells were subcultured in RPMI-1640 medium (10% foetal bovine serum (FBS)), while MRC5 was preserved in Eagle’s Minimum Essential Medium (EMEM, 10% FBS) – all at 37°C, 5% CO2, and 100% relative humidity, for a maximum of 5-10 passages. Cytotoxicity and selectivity studies The cytotoxic effect of four extracts, in addition to doxo- rubicin and camptothecin, was evaluated by the MTT assay, as reported by Alsanosy et al (53) and Abdalla et al (54). Each cell line was cultured separately in 96 wells (3 ×103/well) and incubated with each of the extracts or doxorubicin at a final concentration of 0-100 μg/mL, for 3 days at 37°C overnight (dimethyl sulfoxide (DMSO) 0.1%; n = 3 of three indepen- dent experiments). After 3 days of incubation, the cytotoxic- ity of each extract was evaluated using an MTT assay. MTT was added to each well in culture medium at a concentra- tion of 0.5 mg/mL and incubated for 3 hours at 37°C. The MTT solution was removed and the formazan granules were dissolved by DMSO. The absorbance was read on a mul- tiplate reader (BIORAD, PR 4100, Hercules, CA, USA). The optical density of the purple formazan A550 is proportional to the number of viable cells. The extract concentration caus- ing 50% inhibition (IC50), compared to the control group, 100% cell growth, was estimated using GraphPad Prism. The selectivity index (SI) for the five extracts was calculated by dividing its IC50 for MRC5 cells by the IC50 for MCF7, HT29, or HepG2 cells. Clonogenic assay The clonogenic assay measures tumour cell survival and subsequent proliferative ability after drug exposure (55). The extract (BEP-10) was selected for a further clonogenic test, as it showed the highest selectivity to the normal cell line MRC5. Exponentially growing HepG2 cells in DMEM (supplemented with 10% FBS and 1% penicillin/streptomycin) were seeded in duplicates at a density of 200 cells/well in a 6-well plate and allowed to attach overnight and then exposed to an increasing concentration of BEP-10 (0, 0.75, 1.5, 2.25 µg/mL) for 72 hours. The wells containing the extract were then replaced with fresh media without the extract. The cells were left to grow at 37°C, 5% CO2, and 100% humidity. Daily wells were checked and the cells that form colonies were roughly counted. After 14 days, plates were rinsed in phosphate-buffered saline and fixed with pre-chilled methanol at room temperature for 20 minutes, then stained with 0.5 methylene blue in 1:1 methanol/H2O (v/v) for 10 minutes, washed thoroughly in dH2O, and air dried. Cell colonies were counted and recorded macroscopically. Ethics approval of the study According to the standards of Al-Baha University, all funded project proposals have undergone a critical review followed by approval by relevant scientific research commit- tees before acceptance. Results Phytochemical studies The four plant extracts produced the following yields: BEP-09 (15.3%), BEP-10 (17.3%), BEP-11 (16.2%), and BEP-13 (21.7) from fruits and resins (Fig. 2). FIGURE 2 - Yield % of dry extracts obtained after ethanolic extrac- tion and evaporation of four different selected plants. C; control, ***; p ≤ 0.001. Identification of phytoconstituents using GC-MS Investigation of the GC-MS chromatogram (see the supplementary file) of the fruits of the Miller plant O. ficus- indica (L.) (BEP-10 extract) indicated the presence, mainly, of eight small polar molecules (18) (Tab. 1 and Fig. 3). FIGURE 3 - Structures of chemical constituents identified by gas chromatography-mass spectrometry for the fruits of Opuntia ficus- indica (L.) Miller (BEP-10 extract). In vitro cytotoxicity of Saudi medicinal plants88 © 2024 The Authors. Drug Target Insights - ISSN 1177-3928 - www.aboutscience.eu/dti TABLE 1 - Phytoconstituents identified by GC-MS analysis of the extract of Opuntia ficus-indica (L.) Miller (BEP-10) Compound Formula Molecular weight Peak area (%) Retention time (minutes) Biological activity  (1) �5-(hydroxymethyl)-2- furancarboxaldehyde C6H6O3 126.11 13.2 5.875 Known�to�be�associated�with�antimicrobial� properties�(56)�used�as�an�antifungal�(57)  (2) �1-(4′-Hydroxyphenyl)-2-propanone C9H10O2 150.17 2.52 7.752 Exhibits�a�myriad�of�pharmacological�actions,�such� as�antimicrobial,�antitussive,�antispasmodic,�and� anticancer�properties�(58)  (3) �4-Butyl-phenol� C10H14O 150.22 2.93 8.011 No�significant�report  (4) �1,6-Anhydro-beta-D-glucopyranose C6H10O5 162.14 5.31 8.328 No�significant�report  (5) �Ethylalpha-d-glucopyranoside C8H16O6 208.09 4.60 9.245 Maintenance�and�improvement�of�skin� homeostasis�and�moisturizing�functions�(59)  (6) �Benzeneacetic� acid,� 4-hydroxy-,� methyl�ester C9H10O3 166.06 4.66 9.438 -  (7) �3-Deoxy-d-mannonic�acid C6H12O6 180.16 6.43 9.651 -  (8) �5-(2-Furyl)-3-methyl-penta-2,4- dienoic acid C10H10O3 178.18 3.1 9.843 -  (9) �n-Hydroxydecanoic�acid C16H32O2 256.42 1.89 11.347 As�anti-inflammatory�(60),�cytotoxic�activity�(61) (10) �Hexadecanoic� acid,� 2-hydroxy-1- (hydroxymethyl)ethyl�ester C19H38O4 330.50 3.01 14.656 - (11) �Stigmast-5-en-3-ol C29H50O 414.71 1.54 23.313 Apoptotic�and�antiproliferative�effects�(62) GC-MS�=�gas�chromatography-mass�spectrometry. These molecules represent 73% of the total identified com- pounds. The rest (27%) were the non-polar constituents represented by compounds 9‒11. Therefore, polar mol- ecules constitute >40% of the peak area % relative to the total peak area % of the components that existed in BEP-10 extract. In contrast, non-polar residues represented only 8% of the total peak area % of the components that existed in BEP-10 extract. 5-(Hydroxymethyl)-2-furancarboxaldehyde (1) was found to be the main molecule in BEP-10 extract (13.2%). The other furan derivative (8) was found to have a peak area % of 3.1. Thus, furan derivatives represent the main component of BEP-10 extract (Fig. 4) while phenolic derivatives represented by compounds 2, 3, and 6 came at the second level (peak area % = 10.05) with carbohydrates 4 and 5 (peak area % = 9.91). Cytotoxicity and cell line selectivity studies The four extracts showed a variable IC50 ranging from 0.75 to 19.32 μg/mL. The most active extract was BEP-10 against HepG2 cells, and showed ~4.5-fold selectivity compared to normal MRC5 cells. The selectivity of the extract BEP-10 was greater than that of doxorubicin and camptothecin (Tabs. 2 and 3). FIGURE 4 - Peak areas (%) for the major components of Opuntia ficus-indica (L.) Miller (BEP-10 extract). Alghamdi et al Drug Target Insights 2024; 18: 89 © 2024 The Authors. Published by AboutScience - www.aboutscience.eu TABLE 3 - Selectivity index of the five extracts, doxorubicin, and camptothecin, against normal MRC5 cells Extract MRC5 HT29 HepG2 BEP-09 0.49 0.36 0.57 BEP-10 1.80 0.57 4.47 BEP-11 0.64 0.67 0.77 BEP-12 0.53 0.45 0.91 Doxorubicin 78.57 2.96 2.73 Camptothecin 13.80 0.47 1.55 Clonogenic effect of the extract BEP-10 against HepG2 cells The extract BEP-10 was tested for its possible clonogenic effect against HepG2 liver cancer cells. The extract revealed a dose-dependent clonogenic activity against a dose-depen- dent effect against HepG2 cells that was comparable to the effect of doxorubicin on the same cancer cells (Fig. 5). Discussion Fruits and resins have interesting medicinal uses. In vitro anticancer activity, cell line selectivity, and clonogenicity were considered as a useful trend to scavenge for a useful natural therapeutic agent(s) with putative anticancer property. The extract of O. ficus-indica (fruits; BEP-10) yielded 17.3%. This indicates the high amounts of constituents that are expected to be available in these fruits. The MTT assay of the four extracts showed a vari- able IC50 ranging from 0.75 to 19.32 μg/mL comparable to both standards: doxorubicin (IC50 = 1.40) and camptothecin (IC50 = 1.11), respectively. Fruits appear to be more effec- tive than resins, because they showed lower IC50 values than those produced by standard drugs. These results were consis- tent with those of Castañeda-Arriaga et al (14) who studied the antioxidant effect of this plant and found that its chelat- ing compounds can reduce the harmful effects caused by the most reactive free radical existing immediately. The resulting selectivity (~4.5 fold) of the most active extract BEP-10 against HepG2 cells compared to normal MRC5 cells was found to be higher than that of doxorubicin and camptothecin. The extract BEP-10 was considered for more cytotoxic and mechanistic studies. Selectivity indicates the ability of the extract to have a maximum effect on can- cerous cells and a lesser effect on normal cells. This indicates both its safety and efficacy, and thus it can serve as a promis- ing and useful drug candidate (63). Due to its high selectivity for HepG2 liver cancer cells, the extract BEP-10 was chosen to test its possible clonogenic effect and showed a dose-dependent clonogenic effect com- parable to the effect of doxorubicin in the same cells. The macroscopically counted cell colonies indicate the suppres- sion ability of the active extract, which can be taken as evi- dence to support the preliminary cytotoxicity and selectivity effects. A study by Terzo et al (21) revealed that the O. ficus- indica fruit extract exerted significant antioxidant and anti- inflammatory effects. Correlating the cytotoxic activity of the most promising BEP-10 extract with its phytochemical constituents, GC-MS was performed and different classes of phytoconstituents were detected, including polar molecules (73%) and lipo- philic constituents (27%). These have been reviewed as anti- inflammatory (64), antioxidant (65), and anticancer agents (66). Our results showed that in compound 1, aldehyde 5-(hydroxymethyl)-2-furancarboxaldehyde, the furan deriva- tive was the main compound of the BEP-10 extract. The literature revealed that the medicinal properties of furan include anticancer, antidepressant, antianxiolytic, analgesic, anti-inflammatory, muscle relaxant, antihypertensive, anti- arrhythmic, antimicrobial like antibacterial, antifungal, or antiviral (67), anti-ageing agents, anti-ulcer, antihistaminic, anticholinergic, antiparkinsonian, antidiuretic, and inhibition of sickle cell formation (68). However, the GC-MS chromatogram showed three phe- nolic derivatives (compounds 2, 3, and 6) that were classi- fied as second contents in the BEP-10 extract. These findings were consistent with various studies such as the anticancer (69), anti-trypanosomal activity (70), antileishmanial, anti- inflammatory and antimicrobial activities (71), and anti-neu- roinflammatory and neuroprotective activities (72). Conclusion The study concludes that the O. ficus-indica fruit (BEP-10) is widely distributed in the Al-Baha area and is locally consid- ered a popular fruit. Its extract showed a significant cytotoxic TABLE 2 - Cytotoxic activity of the four extracts, doxorubicin and camptothecin, against three cell lines, and normal fibroblast (MTT 72 hours, IC50, μg/mL ±SD, n = 3) Extract MCF7 HT29 HepG2 Average* IC50 MRC5 BEP-09 6.00±1.61 8.20±0.57 5.20±0.58 6.47 2.94±0.71 BEP-10 1.85±0.73 5.85±0.23 0.75±0.11** 2.82** 3.34±0.41 BEP-11 16.93±0.66 16.07±0.11 14.07±1.04 15.69 10.77±0.54 BEP-12 16.53±0.43 19.32±0.64 9.60±0.82 15.15 8.72±1.56 Doxorubicin 0.07±0.01 1.98±0.10 2.15±0.15 1.40 5.86±0.35 Camptothecin 0.08±0.01 2.50±0.26 0.76±0.07 1.11 1.18±0.10 *Average�cytotoxicity�(IC50)�of�each�extract�against�the�three�cancer�cells.�**p�≤�0.01. In vitro cytotoxicity of Saudi medicinal plants90 © 2024 The Authors. Drug Target Insights - ISSN 1177-3928 - www.aboutscience.eu/dti effect, particularly against HepG2 liver cancer cells with high cell selectivity. Polar and lipophilic phytoconstituents were identified in the plant extract and could contribute to the investigated cytotoxic activity. The furan derivatives that are present as the main compound may play a vital role in the activity stud- ied. Further research is required to obtain the profile of the drug candidate. Acknowledgements This article is a part of the funded project: Investigation of Medicinal Plants with Putative Ocular Effects from Al-Baha Area, Southwestern, Saudi Arabia, Grant number (MOE-BU-4-2020); thus, the funder body is highly acknowl- edged for their financial support. Disclosures Data Availability Statement: All data generated or analysed during this study are available with Dr. Ali as the correspondence author and can be provided upon request. Conflict of interest: The authors declare that they have no compet- ing interests. Funding: The author(s) disclosed receiving the following financial support for the research, authorship, and/or publication of this ar- ticle. This work was supported by the Department of Research and Innovation of the Ministry of Education of Saudi Arabia (grant num- ber: MOE-BU-4-2020). Sponsors did not play a role in the design of the study, the collection and analysis of data, or the preparation of the manuscript. Authors’ contribution: Ali conceived the original idea. Mahadi, Hiadar, and Aimun designed the study. Ashraf, Mohamed EA, Riyad A, and Bassem R conducted the experimental work and collected FIGURE 5 - Colonies of HepG2 cells treated with A) extract BEP-10 (0, 0.75, 1.5, 2.25 µg/mL; n = 2), and B) doxorubicin (0, 2, 4, and 6 µg/mL; n = 2) for 72 hours in 6-well plates followed by a 14-day period of incuba- tion without extract. Bar graph showing x-axis: extract concen- trations (BEP-10) or doxorubicin concentrations; and y-axis: colo- ny number. Results are expres- sed as cell number ± standard deviation of two independent experiments. Alghamdi et al Drug Target Insights 2024; 18: 91 © 2024 The Authors. Published by AboutScience - www.aboutscience.eu the data. Aimun analysed the data and drafted the manuscript. Ali and Asaad, revised it. All approved the final version that was submit- ted. All authors equally contributed to the whole work preparation. All authors approved the version to be published and agreed to be accountable for all aspects of the work. References 1. Meena SS, Lal G, Dubey PN, Meena MD. Medicinal and therapeu- tic uses of Dill (Anethum graveolens L.) – a review. International J Seed Spices. 2019;9(1):14-20. Online 2. Al-Oqail MM, Farshori NN. 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